Absmeier Eva, Rosenberger Leonie, Apelt Luise, Becke Christian, Santos Karine F, Stelzl Ulrich, Wahl Markus C
Laboratory of Structural Biochemistry, Freie Universität Berlin, Takustrasse 6, 14195 Berlin, Germany.
Molecular Interaction Networks, Max-Planck-Institut für Molekulare Genetik, Ihnestrasse 63-73, 14195 Berlin, Germany.
Acta Crystallogr D Biol Crystallogr. 2015 Apr;71(Pt 4):762-71. doi: 10.1107/S1399004715001005. Epub 2015 Mar 26.
The spliceosomal RNA helicase Brr2 is required for the assembly of a catalytically active spliceosome on a messenger RNA precursor. Brr2 exhibits an unusual organization with tandem helicase units, each comprising dual RecA-like domains and a Sec63 homology unit, preceded by a more than 400-residue N-terminal helicase-associated region. Whereas recent crystal structures have provided insights into the molecular architecture and regulation of the Brr2 helicase region, little is known about the structural organization and function of its N-terminal part. Here, a near-atomic resolution crystal structure of a PWI-like domain that resides in the N-terminal region of Chaetomium thermophilum Brr2 is presented. CD spectroscopic studies suggested that this domain is conserved in the yeast and human Brr2 orthologues. Although canonical PWI domains act as low-specificity nucleic acid-binding domains, no significant affinity of the unusual PWI domain of Brr2 for a broad spectrum of DNAs and RNAs was detected in band-shift assays. Consistently, the C. thermophilum Brr2 PWI-like domain, in the conformation seen in the present crystal structure, lacks an expanded positively charged surface patch as observed in at least one canonical, nucleic acid-binding PWI domain. Instead, in a comprehensive yeast two-hybrid screen against human spliceosomal proteins, fragments of the N-terminal region of human Brr2 were found to interact with several other spliceosomal proteins. At least one of these interactions, with the Prp19 complex protein SPF27, depended on the presence of the PWI-like domain. The results suggest that the N-terminal region of Brr2 serves as a versatile protein-protein interaction platform in the spliceosome and that some interactions require or are reinforced by the PWI-like domain.
剪接体RNA解旋酶Brr2是在信使RNA前体上组装具有催化活性的剪接体所必需的。Brr2具有不同寻常的结构，包含串联的解旋酶单元，每个单元由两个类RecA结构域和一个Sec63同源单元组成，其前面还有一个超过400个残基的N端解旋酶相关区域。尽管最近的晶体结构已经为Brr2解旋酶区域的分子结构和调控提供了见解，但对其N端部分的结构组织和功能却知之甚少。本文展示了嗜热毛壳菌Brr2 N端区域中一个类PWI结构域的近原子分辨率晶体结构。圆二色光谱研究表明，该结构域在酵母和人类Brr2直系同源物中是保守的。虽然典型的PWI结构域作为低特异性核酸结合结构域起作用，但在带移实验中未检测到Brr2异常PWI结构域对多种DNA和RNA有明显亲和力。同样，在目前晶体结构中所见构象的嗜热栖热菌Brr2类PWI结构域，缺乏至少一个典型核酸结合PWI结构域中观察到的扩展正电荷表面斑块。相反，在针对人类剪接体蛋白的全面酵母双杂交筛选中，发现人类Brr2 N端区域的片段与其他几种剪接体蛋白相互作用。这些相互作用中至少有一个与Prp19复合蛋白SPF27的相互作用，依赖于类PWI结构域的存在。结果表明，Brr2的N端区域在剪接体中作为一个多功能的蛋白质-蛋白质相互作用平台，并且一些相互作用需要类PWI结构域或被其加强。
